The three-dimensional structure of the vnd/NK-2 homeodomain bound to an uncommon 18 base-pair DNA consensus segment that contains 5'-CAAGTG-3' has been analyzed by NMR spectroscopy. The interactions responsible for the nucleotide sequence-specific binding have been identified, where tyrosine in position 54 previously was shown to be the major determinant of the unusual consensus nucleotide sequence. Mutation analysis was carried out on for positions (35, 52, 54, and 56) in the homeodomain. Mutation of position 54 from tyrosine to methionine results in a decrease of one order of magnitude in the binding affinity of the homeodomain for the DNA consensus sequence. Mutation of position 35 from alanine to threonine results in a homeodomain that is unable to adopt a folded conformation free in solution at temperatures down to -5 degrees Celsius. The alanine to threonine mutant homeodomain specifically binds to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower that that of the wild-type homeodomain. Although the three-dimensional structure of the mutant in the DNA bound state shows characteristic helix-turn-helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i, i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the mutant this amide proton hydrogen bond is altered and the structure of the protein in the region of helix-II is distorted relative to that of the wild-type analog. .